We found that the glgP gene of this organism was induced with maltose, and the gene likely constituted an operon with the upstream gene malQ. This putative operon was negatively regulated with the malR gene located upstream from the malQ gene and a MalR-binding consensus sequence was found upstream of the malQ gene. S. mutans is not able to catabolize starch. However, this organism utilizes maltose degraded from starch in the presence of saliva amylase. Therefore, we hypothesized that the MalQ/GlgP system may participate in the metabolism of starch-degradation products.The malQ or glgP gene overexpressed His-tagged proteins with the plasmid pBAD/HisA. S. mutans malQ and/or glgP mutants were also constructed. Purified proteins were assayed for glucose-releasing and phosphorylase activities with appropriate buffers containing maltose, maltotriose, maltodextrin, or amylodextrin as a substrate, and were photometrically assayed with a glucose-6-phosphate dehydrogenase–NADP system.A DNA fragment amplified from the Purified MalQ protein released glucose from maltose and maltotriose but did not from either maltodextrin or amylodextrin. The purified GlgP protein did not exhibit a phosphorylase reaction with maltose or maltotriose but generated glucose-1-phosphate from maltodextrin and amylodextrin. However, the GlgP protein released glucose-1-phosphate from maltose and maltotriose in the presence of the MalQ protein. In addition, the MalQ enzyme activity with maltose released not only glucose but also produced maltooligosaccharides as substrates for the GlgP protein.malQ gene encodes 4-α-glucanotransferase but not α-1,4-glucosidase activity. The malQ mutant could not grow in the presence of maltose as a carbon source, which suggests that the malQ gene is essential for the utilization of starch-degradation products.These results suggest that the  Streptococcus mutans is a major etiologic agent of human dental caries    with resectively . Therefohorylase  are locaglgP gene region and found a potential promoter-like sequence located in the 114-bp intergenic region between the malQ and upstream malR genes. In addition, a MalR-binding consensus sequence reported in Streptococcus pyogenes and malXFGK operons respectively encoding IIABCmaltose of the phosphoenolpyruvate-dependent maltose phosphotransferase system (PTS) (phsG were located on another part of the chromosome (glgBCDA), although a candidate for a debranching enzyme was not present in this gene cluster.However, we recently reanalyzed the genome sequence around the pyogenes was deteromosome  as a cluS. mutans could not grow with starch as a direct carbon source, but grew well with maltose or maltooligosaccharides derived from starch in the presence of a small amount of saliva. Therefore, we presumed that the malQ/glgP genes may be involved in the catabolism of maltose or maltooligosaccharides derived from the starch supplied in food in the oral environment rather than the degradation of intracellular glycogen-like polysaccharides. This suggested that this organism, as well as other oral streptococci , zJU1 , zKU1 (glgP), and aeU1 . Streptococci were maintained on Todd-Hewitt (TH) broth/agar plates with or without appropriate antibiotics. Escherichia coli strain TOP10 was used as a host with the vector pBAD/HisA for the expression of cloned genes as N-terminal histidine-tagged proteins.re UA159  and its malQ and glgP genes in strain UA159 were amplified with the primer sets exmalQ5/malQ32 and expglg51/expglg31, respectively. Amplified fragments were purified, digested with XhoI, and subcloned into XhoI/PvuII-double-digested pBAD/HisA. The fragment (fk) was constructed by inserting the kanamycin-resistant gene cassette into the BamHI site in the middle of the malR gene, which was subcloned into a pUC-vector . Target regions containing primer-annealing sites that were used in this technique are noted in S. mutans UA159.The polymerase chain reaction (PCR) primers used in this study are listed in C-vector  13). Sp. SpmalQ C-vector  was emplS. mutans strains and mutants were grown in 10-ml BTR-Sugar broth . Cells were harvested, washed, disrupted with a Tissue Lyser  with 0.2 mm diameter ceramic beads, and subjected to centrifugation  with a microfuge to remove undisrupted cells. Four hundred microliters of supernatant fluid was obtained as a crude extract sample and its protein concentration was determined with DC protein assay reagents . SDS-PAGE was run with 2.5 µg of samples for Quick-CBB-PLUS  staining and 0.4 µg of samples were used for Western blot analysis with previously prepared anti-GlgP serum  as described previously  as described previously, and the protein concentrations of the purified proteins were determined with DC protein assay reagents. Purified samples were frozen at −20°C for later enzyme assays. The enzyme activities of these samples were stable for at least 8 months.2, and sugar substrate. The sugar substrates used in the enzyme reaction were 1% maltose, maltotriose, maltodextrin, and 0.8% amylodextrin . Both glucose-releasing and phosphorylase reactions were not linear as a function of time. Therefore, the incubation time was fixed at 10 min at 37°C followed by 5-min inactivation at 95°C. Glucose and G1P were formed in the reaction mixtures containing MalQ and GlgP proteins, respectively. Glucose or G1P in aliquots were spectrophotometrically determined by the end-point method of absorbance changes at 340 nm as a result of the amount of generated NADPH in the assay mixtures as recommended by the supplier  of the enzymes. Glucose and G1P assays were started with the addition of 2 IU of hexokinase and phosphoglucomutase, respectively.Enzyme reactions both for the MalQ and GlgP proteins were performed in 50-µl reaction mixtures within a thin-wall-PCR tube with starch-degradation products as substrates. The standard enzyme reaction mixtures for glucose- and glucose-1-phosphate (G1P) assays were composed as recommended by the supplier  of enzymes used in the assay system. The enzyme reaction mixture contained 0.7–1.0 µg of purified MalQ protein or 2.5–3.2 µg of purified GlgP protein in addition to 50 mM potassium phosphate (pH 7), 0.5 mM MgClS. mutans strains and mutants in BTR-sugar broth was measured at an optical density (OD) of 660 nm with the Ultrospec 500 pro spectrophotometer . Values of OD660nm were recorded at 1-hour intervals following the inoculation of cultures into screw-capped glass tubes containing BTR-sugar broth. Sugars included maltose, maltotriose, maltodextrin, and amylodextrin as starch-degradation products, other disaccharides, as well as glucose as a control.The growth of p-anisaldehyde solution containing ethanol/sulfuric acid/acetic acid/p-methoxybenzaldehyde (68: 2.5: 1.2: 1.9) and heated on a hotplate  for 40 min at a dial setting of 4.5.The reaction products liberated in the MalQ enzyme reaction mixture described above incubated with 1% maltose as a substrate for 100 min were characterized with respect to molecular size by thin-layer ascending chromatography on a 0.25-mm silica-gel-coated glass plate  employing a solvent system of 1 M lactic acid/acetone/2-propanol  . CarbohyThe MalQ or GlgP protein was expressed as a His-tagged protein, purified, and the enzyme activities were determined as described in ‘Materials and methods’ section. The MalQ protein exhibited glucose-releasing activities with maltose and maltotriose and low activity with maltodextrin but not with amylodextrin. The GlgP protein did not exhibit phosphorylase activities with maltose and maltotriose but did with maltodextrin and amylodextrin . MoreovemalR-mutant fkU1 and aeU1 , which was used to transform S. mutans strain UA159, are also indicated in malR gene. We confirmed the constitutive expression of the GlgP protein with glucose-grown cells in contrast to the absence of GlgP expression in glucose-grown UA159 or zJU1 cells as indicated in glgP internal fragment was replaced with the streptococcal kanamycin-resistant gene kanr . The finding that this mutant could not grow with maltotriose or maltodextrin (data not shown) in addition to maltose suggests that the malQ gene is very likely essential for the utilization of starch-degradation products, but does not participate in the energy metabolism of the other sugars mentioned above. The GlgP mutant zKU1 grew well with glucose similar to UA159. However, the growth of this mutant in the presence of maltose as the sole carbon source was slightly less than that when glucose was used as a carbon source. The reason for this phenomenon will be discussed below. These results together with the observation that the GlgP protein had no phosphorylase activities for maltose or maltotriose described above suggest that the glgP gene is not essential for, but somewhat influences the utilization of maltose and maltotriose.Parental strain UA159 grew well with either glucose or maltose as a carbon source b. In conmalQ and glgP genes were likely co-transcribed because the malQ mutant zJU1 was GlgP negative as described above. Therefore, these two proteins may work together on their substrates or products in vivo. Glucose-releasing and phosphorylase activities on starch-degradation products were determined in the presence of both of the purified MalQ and GlgP proteins      activity in partially purified preparations obtained from S. mutans strain 6715-49 (this strain is currently classified as S. sobrinus) cells grown with maltose as the main carbon source, and indicated that maltose participated as a donor as well as an acceptor for the enzyme activity  , Clostrim  , and E. )  . 4-α-Glu)  , 1920)Streptoco maltose . How couactivity . S. mutaS. mutans cells may be maltose-6-phosphate, which is taken up into cells predominantly through IIABCmaltose  of the phosphoenolpyruvate-dependent maltose PTS in S. mutans in Enterococcus faecalis hydrolase protein pairs specific for these disaccharides that induce the corresponding pairs  of PTS was not proximal to the corresponding sugar phosphate hydrolase gene, which may be compatible with a malT-mapP gene arrangement in E. faecalis.The ng pairs –27. In cS. mutans cells, starch-degradation products including maltose as the smallest molecule would be catabolized in concert with the malQ, glgP, and amy(glgP mutant zKU1 was suppressed when maltose was used as the sole carbon source (Even if such a hydrolase for maltose is absent in  and amy gene pro and amy. However and amy in the p and amy. The gron source b. This mS. mutans alone was unable to ferment starch (S. mutans.t starch a similart starch . Howevert starch , which dmalQ gene involved in glucose-releasing activity is essential for utilizing starch-degradation products similar to the malP maltose phosphorylase gene in E. faecalis(malQ gene may be a target for controlling S. mutans in oral environments.We now report that the purified MalQ and GlgP proteins exhibited glucose release from maltose/maltotriose and phosphorylase activity from maltodextrin/amylodextrin, respectively. In addition, the faecalis. Therefo


Patients with platinum-sensitive recurrent ovarian cancer are a heterogeneous group, and it is not possible to accurately predict the progression-free survival (PFS) in these patients. We developed and validated a nomogram to help improve prediction of PFS in patients treated with platinum-based chemotherapy.n=955) from the CALYPSO trial and validated in the AGO-OVAR 2.5 Study (n=340). The proportional-hazards model (nomogram) was based on pre-treatment characteristics.The nomogram was developed in a training cohort (The nomogram had a concordance index (C-index) of 0.645. Significant predictors were tumour size platinum-chemotherapy-free interval, CA-125, number of organ metastatic sites and white blood count. When the nomogram was applied without CA-125 , the C-indices were 0.624 (training) and 0.594 . When classification was based only on the platinum-chemotherapy-free interval, the indices were 0.571 (training) and 0.560 . The calibration plot in the validation cohort based on four predictors (without CA-125) suggested good agreement between actual and nomogram-predicted 12-month PFS probabilities.This nomogram, using five pre-treatment characteristics, improves prediction of PFS in patients with platinum-sensitive ovarian cancer having platinum-based chemotherapy. It will be useful for the design and stratification of patients in clinical trials and also for counselling patients. Ovarian cancer is a common cause of cancer death in women from developed countries  were retained. The variables were assigned points on a scale for constructing the nomogram.The primary end point was PFS by the RECIST  criteria , stratifPatients were grouped by quartile on the basis of the predicted probability of PFS. The first quartile (score⩽30) formed the good-prognosis group. The middle two quartiles (31⩽score⩽68) were combined to form the intermediate-prognosis group. The final quartile (score⩾69) formed the poor-prognosis group.vs predicted PFS for each of the three prognosis groups. Tests of goodness-of-fit were used to compare observed and predicted PFS over deciles of the risk score. A significant P-value for this statistic shows lack of calibration of the model   statistic  was calculated with the model refitted 200 times by bootstrap resampling in the training cohort. The C-index estimates the proportion of all pairwise combinations of patients whose PFS times are ordered, such that the patient with the longest predicted PFS was the one who actually lived longer (discrimination)   . Women iIn the training cohort, the median follow-up duration was 17.5 months (range 0–37.5). The proportion of women surviving at 12 months was 44.3% , 40.9–47.6%) . In multA point scale was used to assign points to these five variables in the nomogram based on the multivariable model . The sumP<0.0001).The model showed good discrimination . The good-prognosis group (score⩽30) comprises 229 patients (24%) with a median PFS of 16.1 months  and 1-year PFS of 64.6% . The intermediate-prognosis group (31⩽score⩽68) comprises 502 patients (53%) with a median PFS of 11.8 months  and 1-year PFS of 45.6% . The poor-prognosis group (score⩾69) comprises 224 patients (23%) with a median PFS of 9.0 months  and 1-year PFS of 20.9% . P<0.0001).When CA-125 was omitted from the multivariable model, the bootstrap-corrected C-index for the nomogram decreased to 0.631 . The good-prognosis (222 patients), intermediate-prognosis (486 patients) and poor-prognosis (247 patients) groups had median PFS of 17.4, 11.7 and 9.1 months, respectively . Information on CA-125 was unavailable from this cohort. The C-index, when the nomogram (without CA-125) was applied, was 0.594.P=0.008). The good-prognosis group had a median PFS of 9.1 months  and 1-year PFS of 32.2% . The intermediate-prognosis group had a median PFS of 7.8 months  and 1-year PFS of 23.4% . The poor-prognosis group had a median PFS of 6.4 months  and 1-year PFS of 12.1% .Only 53 patients (15.6%) were classified with this nomogram as having good prognosis and 221 (65.0%) and 66 (19.4%) patients were classified as having intermediate and poor prognosis, respectively. vs predicted PFS for each of the three prognosis groups (χ2=11.41 (8 degrees of freedom), P=0.18).The calibration plot of the actual s groups  indicatevs 9.2 months, log-rank P<0.0001). The C-index was 0.571.In the training cohort, complete ‘platinum-sensitive’ patients  had a longer median PFS than partial ‘platinum-sensitive’ patients  . The C-index was to 0.560.In the validation cohort, complete ‘platinum-sensitive’ patients also had a longer median PFS than partial ‘platinum-sensitive’ patients . In both the training and validation data sets, the platinum-chemotherapy-free interval alone performed poorly in discriminating a patient's prognosis . Prognostic stratification is improved with the combination of five predictors over the platinum-chemotherapy-free interval alone.In the absence of a cancer staging system for patients with recurrent ovarian cancer, the nomogram could be used to stratify patients on the basis of our risk-score cutoff points in randomised trials. A consistent definition of risk based on this nomogram will allow selection of a more homogeneous cohort of patients, ensure better balance of important prognostic factors in various arms of a randomised study and improve international collaboration in clinical trials through adherence to an identical prognostic classification.The nomogram can also be used to enrich clinical trials by targeting specific risk groups. For example, only poor-prognosis patients could be recruited to trials of novel approaches to treatment. In contrast to ‘all comers’ studies, ‘enrichment’ trials will have more power to detect the treatment effect and substantially reduce the patient accrual needed. Such an approach is also ethically desirable because it can minimise patients’ exposure to experimental treatment of unproven benefits.This nomogram is also a pragmatic tool that uses readily available clinical information to provide simple prognostic information for oncologists and patients from complex statistical estimates. Most patients with advanced cancer would like information about their prognosis . Recent The nomogram improves our understanding of the relationship between disease burden and PFS. It has been argued that conventional imaging techniques do not easily detect peritoneal carcinomatosis in patients with recurrent ovarian cancer (This study also supports the role of the white blood cell count as a potential marker of inflammation and prognosis. Although the reason for the association between elevated white cell count and PFS is uncertain, several studies have reported that it was an adverse prognostic factor in patients with recurrent ovarian cancer  and are This nomogram has a number of limitations. First, it was developed and validated on data from highly selected patients who were enrolled in two large phase III trials. Its applicability to patient groups outside clinical trial settings and patients treated with non-platinum treatments (In conclusion, we have developed a robust prognostic nomogram to predict PFS in patients with platinum-sensitive recurrent ovarian cancer undergoing platinum-based chemotherapy. This nomogram represents an improvement in prognostication over the platinum-free chemotherapy interval alone. This tool can facilitate the design and implementation of future collaborative randomised and non-randomised clinical trials. It also represents an important first step towards providing prognostic information for patients with this life-threatening illness to guide treatment selection.


There is some research on taste disorder/hyposensitivity in special groups such as the elderly or patients presenting with specific taste problems, however few studies have been conducted among young populations. The objectives of this study were to estimate the prevalence of taste hyposensitivity and to investigate the relationship between taste hyposensitivity and oral health status in Japanese schoolchildren.Subjects were 237 primary and 112 junior high school students in Saitama Prefecture, Japan. In total, 349  students aged 6–15 years participated in the study. Oral examinations and whole-mouth taste tests using four tastes  solutions were conducted on the subjects. A subject who could not recognize the taste of the solution was defined as demonstrating hyposensitivity.th-9th grade subjects. Furthermore, the prevalence of bitter-taste hyposensitivity was significantly higher in males than females among 1st-3rd graders.Hyposensitivity was observed in 6.3% of all subjects for sweet-taste, 14.3% for salt-taste, 20.9% for sour-taste and 6.0% for bitter-taste. The prevalence of sweet, sour and bitter-taste hyposensitivity decreased as the subjects’ grade advanced. In contrast, the prevalence of salt-taste hyposensitivity increased in 7Taste hyposensitivity had little association with oral health status, such as decayed teeth, filled teeth, dental plaque, gingival status and tongue coating.In this study, taste hyposensitivity was observed in 6.0%-20.9% of the students. There was little association between taste hyposensitivity and oral health status. The current study implies that the factors affecting the taste hyposensitivity in children may different from those in the elderly. Therefore it is necessary to further investigate the causes of taste hyposensitivity among younger generation. Accordiery year . HoweverVarious taste tests exist with differing advantages and disadvantages. For example, the taste disc method is a precise tool but takes a long time to perform per person . TherefoThe objectives of this study were to estimate the prevalence of taste hyposensitivity using the whole-mouth taste test for four basic tastes  and to investigate relationships between taste hyposensitivity and oral health status in schoolchildren.st-6th grade: boys: 121, girls: 116) and 112 junior high  schoolchildren aged 6–15 years in Saitama Prefecture, Japan. In total, 349 students  participated in the research. Students were 1st to 9th grade  schoolchildren. The research was implemented as part of school health promotion activities, therefore basically all students participated in the activity except for those who were absent. The information about this study was explained to all the students, and the consent was received from the subjects who agreed to participate in the study. The research was conducted in 2009 and the study proposal was approved by the Tokyo Medical and Dental University Ethics Committee .Subjects in this research were 237 primary . Gingival condition was investigated with a gingival status score ranging from 0 to 2 .et al.[The tongue coating area or thickness was evaluated visually with the criteria described by Oho et al.. The areTaste tests were performed using a whole-mouth method . A 3% suA total of 1 mL of taste test solution was placed on the center of the tongue dorsum with a syringe. Immediately following the test, the students were asked to respond to whether or not they identified the taste tested. Between taste tests, the students rinsed their mouths with distilled water in order to eliminate the influence of the previous taste.st-3rd, 4th-6th and 7th-9th) by grade.Students were divided into three groups  and p-values less than 0.05 were considered to be statistically significant.Associations between taste hyposensitivity and oral health status were analyzed by Χst-3rd graders (p < 0.05). A statistically significant difference in the distribution of gingivitis by gender was detected among 4 th-6th graders (p < 0.05). No significant differences were observed in other oral health items between boys and girls.Table st-3rd grade), 4.8% (4th-6th grade), 3.6% (7th-9th grade) and 6.3% . The prevalence of salt-taste hyposensitivity were 16.2% (1st-3rd grade), 8.7% (4th-6th grade), 18.8% (7th-9th grade) and 14.3% . The prevalence of sour-taste hyposensitivity were 28.8% (1st-3rd grade), 20.6% (4th-6th grade), 13.2% (7th-9th grade) and 20.9% . The prevalence of bitter-taste hyposensitivity were 16.2% (1st-3rd grade), 2.4% (4th-6th grade), 0% (7th-9th grade) and 6.0% .Table th-6th grade and increased in 7th-9th grade (p < 0.05).The prevalence of sweet-, sour- and bitter-taste hyposensitivity decreased as the schoolchildren’s grade advanced, while the prevalence of salt-taste hyposensitivity decreased up to 4In total, there was a significant gender difference in the prevalence of salt- or bitter-taste hyposensitivity (p < 0.05). There were no significant differences between boys and girls in sweet- and sour-taste.st-3rd and 7th-9th graders (p < 0.05) and sour-taste hyposensitivity between 1st-3rd graders and 7th-9th graders (p < 0.05). In the prevalence of bitter-taste hyposensitivity, there were significant differences between 1st-3rd graders and 4th-6th graders (p < 0.01), and between 1st-3rd graders and 7th-9th graders (p < 0.01).As for differences by grade Table , there wth-6th graders (p < 0.05) in this study.As presented in Table Taste disorder was classified as the condition that expressed the impairment of taste sensation ,9,10. InAccording to the another study, it was reported that the taste threshold declines in most of junior high school students . Taste sth-6th graders and the highest in 7th-9th graders. The higher prevalence of salt-taste hyposensitivity found in junior high school students might be related to the diet, especially the amount of salt intake [However, salt-taste showed a different tendency. The prevalence of salt-taste hyposensitivity was the lowest in 4t intake . Our fort intake . HoweverMany studies report that there is an association between taste sensitivity and zinc ,16-19. TAn analysis by gender found that the prevalence of bitter-taste hyposensitivity was higher in males than females. This result was similar to that in previous research, which reported a higher prevalence of women’s bitter-taste sensitivity than men .et al. examined the taste of 18–31 years old university students by using the taste-disc method. The study revealed that 24.8% of subjects had a taste disorder and the alteration of taste sensitivity was related to dry mouth, tongue coating and damage or loss of taste buds [Several studies reported that oral health status was associated with taste hyposensitivity in the elderly -24. Satoste buds .However, a slight association was only observed between taste hyposensitivity and area of tongue coating in this study targeted school children. Further scrutiny of the relationship of taste disorder/hyposensitivity with oral health status will be necessary.st-3rd grade presented sour or bitter-taste hyposensitivity. These generations of students might not recognize the sour- and bitter-taste correctly. One of the plausible reasons for this finding is that taste sensation does not have enough sensitivity, because the taste sensation may be not completely developed yet at this age. However this has not been demonstrated. It is necessary therefore to confirm the hypothesis biologically and continue research by performing sour- or bitter-taste test for younger grades of students.In this study, relatively large number of students in the 1st-9th grade students has not yet been established. The taste disc method is used to diagnose a taste disorder in individuals in a clinical environment, but it takes approximately 1 hour per person [There are various kinds of methods for taste tests, but which method is adequate for 1r person . On the r person . The whoTaste disorder/hyposensitivity is not the only dental problem. It also greatly influences dietary behavior, nutrition and systemic health ,27. As aWe conducted taste tests for four tastes using the whole mouth method, in schoolchildren aged 6–15 years. Taste hyposensitivity was observed in 6.0-20.9% of children. There was little association between taste hyposensitivity and oral health status. The current study implies that the factors affecting the taste hyposensitivity in children may different from those in the elderly. Therefore it is necessary to further investigate the causes of taste hyposensitivity among younger generation.The authors declare that they have no competing interests.MO carried out the conception and design of the study, data acquisition, data analysis and draft the manuscript. MU participated in the conception and design of the study, data acquisition. TZ participated in the data acquisition. YK conceived of the study, and participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/14/36/prepub


They found that MI increased the heart weight and myocyte size which was restored by cAng-(1-7) to sham levels. In addition, cAng-(1-7) lowered left ventricular end-diastolic pressure and improved endothelial function. The second strategy discussed in this special issue was the Ang-(1-7) included in hydroxypropyl In this special issue, the role of the ACE2/Ang-(1-7)/Mas axis in the cardiovascular function was extensively discussed in review articles and new findings regarding this axis were presented. E. R. M. Gomes et al. revised the actual knowledge about Ang-(1-7)-mediated signaling in cardiac cells, as well as the discoveries made in cardiomyocyte physiology through the use of genetic approaches. Interestingly, M. Poglitsch et al. reported significant differences in the conversion rates of recombinant human and recombinant murine ACE2  for diverse natural peptide substrates in plasma samples. They found species-specific differences in substrate specificities, probably leading to functional differences in the alternative axis of the RAS. Specially, in contrast to rhACE2, rmACE2 is substantially less potent in transforming Ang-(1-10) to Ang-(1-9). Of note, this enzymatic pathway was reviewed by M. P. Ocaranza and J. E. Jalil. They presented current experimental evidence suggesting that activation of the ACE2/Ang-(1-9) pathway might protect the heart and vessels from adverse cardiovascular remodeling in hypertension and in heart failure. In addition to the cardiovascular system, some insights were given in the renal system in this special issue. S. V. B. Pinheiro and A. C. Simões e Silva highlighted the current understanding of the ACE2/Ang-(1-7)/Mas axis in renal physiology and in the pathogenesis of primary hypertension and chronic kidney disease. Also, L. C. Barroso et al. presented some data demonstrating the renal effects of acute administration of AVE 0991 in a murine model of renal ischemia/reperfusion. Administration of AVE 0991 promoted renoprotective effects evidenced by improvement of renal function, decreased tissue injury, prevention of local and remote leukocyte infiltration, and release of the chemokine CXCL1. In the past few years, the understanding of the RAS has improved significantly. This is not only a consequence of the identification of novel members, but also due to advances in the knowledge concerning the role of these components in many tissues and cells , 6. Here2 receptors appear to be involved in the Ang-(1-7) effects [2 in selected tissues such as heart has been suggested as a putative mechanism for Ang-(1-7) actions [2 receptors. S. Bosnyak et al. reported that the hypotensive effect of Ang-(1-7) was dependent on the background dose of candesartan and that this effect was reversed by AT2 receptor blockade. In aged rats, the depressor effect of Ang-(1-7) was evident but was inhibited by either AT2 or Mas receptors blockade. Furthermore, E. S. Jones et al. reported that AT2 receptor stimulation does not significantly influence the antihypertensive effect of chronic AT1 receptor blockade but plays a role in the regulation of vascular structure, as well as in cardiac perivascular fibrosis. In certain circumstances and in some tissues, AThe Ang-1- effects  actions . In thisThus, we hope that this special issue is able to make a picture of the role of the ACE2/Ang-(1-7)/Mas axis and of the potential therapeutic of this concept for treating cardiovascular and related diseases. Our expectation is that this special issue might serve as an important reference to scientists and physicians to update their knowledge about this contemporary theme. Anderson J. FerreiraAnderson J. FerreiraRobson A. S. SantosRobson A. S. SantosMohan K. RaizadaMohan K. Raizada


Blood samples from 260 female patients were collected one week following a CS for the detection of p53 gene polymorphisms using a molecular beacon-coupled quantitative polymerase chain reaction technique. Patients had follow-ups for 12–18 months to observe the scar formation. From these observations, the relationship between the p53 codon-72 polymorphisms and hypertrophic scar formation occurrence was investigated. Among the patients with the CCC/CCC genotype, nine patients had hypertrophic scars and 46 patients showed normal healing, which is a ratio of 0.19. However, the follow-up investigations indicated that the presence of a homozygous or heterozygous C-to-G alteration at the codon-72 site in gene p53 resulted in 13 patients with hypertrophic scars and 192 patients with normal healing, which is a ratio of 0.07. Therefore, these results indicate that patients with the CCC/CCC genotype had a higher risk of developing hypertrophic scars compared with that for patients with the CCC/CGC or CGC/CGC genotypes.The aim of the present study was to determine the relationship between  Abnormal scars, including keloid and hypertrophic scars, are characterized by abnormal fibroproliferation and increased extracellular matrix production ,2. In prt cancer . Abnorma keloids . In addiearchers –9. HowevIn total, 260 female patients with an average age of 29.4 years (ranging between 20 and 39 years) and of the same ethnicity were selected from the Nanfang Hospital of Southern Medical University  for the study. The patients did not have recorded histories of pathological scar formation or benign or malignant tumors. Blood samples were collected one week following the CS. Informed consent was obtained from each patient prior to enrollment in the study. All specimens were obtained following informed consent and procedures were conducted in accordance with the Ethical Standards of the Declaration of Helsinki . Writtenp53 codon-72. The 5′ end of the CCC and CGC probes were labeled with carboxyfluorescein (Fam) and hexachlorofluorescein (Hex), respectively , the 3′ end was conjugated with a quenching complex . The sequences of the probes were as follows: MBC, 5′-Fam-ATGCAGCCCCcCGTGGCCCCTGCAT-BHQ-1; and MBG, 5′-Hex-ATGCAGCCCCgCGTGGCCCCTGCAT -BHQ-1. Two primers, designed using Primer 5 software  and synthesized by Shanghai Genechem Co., Ltd. , were used to amplify the p53 380-bp fragment in exon 4. The primer sequences were as follows: Forward, TPF, 5′-GACCTGGTCCTCTGACTGCT-3′; and reverse, TPR, 5′-GATACGGCCAGGCATTGAAG-3′.Genomic DNA was extracted from 2 ml blood samples collected from the peripheral veins of each patient. A pair of molecular beacon probes were designed and labeled with fluorescent dyes to detect the single nucleotide polymorphism (SNP) of For PCR, 0.5 μl of each primer, 1 μl genomic DNA, 0.2 μl Fam-C primer and 0.2 μl Hex-G primer were mixed and then diluted with DNase- and RNase-free water to provide a total reaction volume of 20 μl. The reaction was performed with a Stratagene Mx3000P qPCR system . The amplified gene products were sent to Sangon Biotech Co., Ltd.  for sequencing. PCR products were detected by electrophoresis.Nested PCR was preformed to obtain the p53 380-bp fragment in exon 4. Briefly, DNA extracted from peripheral blood was amplified by the first set of primers. The product obtained was then used as the template for a second amplification with primers of TPF and TPR. Finally, the PCR products from the second amplication were analyzed by agarose gel electrophoresis.Follow-up investigations were performed for a period of 12–18 months. During this period, patients were required to return to the hospital at any time when a fibroproliferative scar appeared to grow beyond the confines of the original wound. Patients were also required to report to the hospital if symptoms, including skin erythema, pruritus or pain, developed at the site of surgical incision.p53 gene polymorphisms and the occurrence of hypertrophic scars was evaluated by a χ2 test and a Student’s t-test. Moreover, the significant association between p53 gene polymorphisms and hypertrophic scars was also evaluated by a corrected χ2 test. SPSS 13.0  was used to carry out statistical analysis. P<0.05 was considered to indicate a statistically significant difference.The relationship between the Peripheral venous blood samples were collected from 260 patients in the study. A total of 249 patients underwent the lower abdominal CS  via a trThe products of nested PCR were detected following an electrophoresis reaction. As shown in p53 codon-72 polymorphisms, fluorescence qPCR was performed. As shown in To determine the gene p53 resulted in 13 patients with hypertrophic scars and 192 patients with normal healing, which is a ratio of 0.07 , which is associated with various types of cancers, is the most extensively studied SNP in the p53 gene. Specific studies have reported that the p53 codon-72 CGT/CCT SNP is also associated with abnormal scar susceptibility; however, this remains controversial. Wang et al (p53 in 54 patients with keloids and 30 patients with hypertrophic scars using restriction fragment length polymorphism. The study observed that the frequencies of the Pro- and Arg-encoding alleles in the hypertrophic scar patients deviated significantly from those in the normal controls. However, there was no significant difference between the keloid patients and healthy individuals. Zhuo et al (et al (p53 codon-72 polymorphism between keloid patients and healthy controls, but that the Arg/Arg genotype may affect the formation of keloids in the shoulder and back.Possible mechanisms underlying the pathogenesis of hypertrophic scars include excessive inflammation, excessive angiogenesis, abnormal growth factor levels and delayed apoptosis of fibrotic myofibroblasts due to alterations to the p53 gene –14. In pp53 gene . p53 mutp53 gene . The p53uo et al  analyzedl (et al  hypothesp53 codon-72 CCC/CCC genotype may result in keloid susceptibility in the Guangdong district (p53 codon-72 polymorphisms may change gene expression, resulting in changes to p53 function, including activating gene transcription, inducing apoptosis and inhibiting cell transformation (A study has shown that the district . Furtherdistrict . In addip53 codon-72 CCC/CCC genotype was 163.6/1,000. Patients with the p53 codon-72 CCC/CCC genotype were the susceptible population, whose relative risk of hypertrophic scars was 2.8896-fold higher than that of the other individuals. The results of the present study indicate that patients with the CCC/CCC genotype had a higher risk of developing hypertrophic scars compared with that of patients with CCC/CGC or CGC/CGC genotypes.In the present study, the PPV of abnormal scars following CS in patients with the


Ca+2-mediated signalling processes are usually carried out through well characterized proteins like calmodulin (CaM) and other Ca+2 binding proteins (CaBP). These proteins interact with different targets and activate it by bringing conformational changes. Majority of the EF-hand proteins in Caenorhabditis contain Ca+2 binding motifs. Here, we have performed homology modelling of CaM-like proteins using the crystal structure of Drosophila melanogaster CaM as a template. Molecular docking was applied to explore the binding mechanism of CaM-like proteins and IQ1 motif which is a ∼25 residues and conform to the consensus sequence QXXXRXXXX to serve as a binding site for different EF hand proteins. We made an attempt to identify all the EF-hand  containing proteins and their Ca+2 binding affinity in Caenorhabditis by analysing the complete genome sequence. Docking studies revealed that F165, F169, L29, E33, F44, L57, M61, M96, M97, M108, G65, V115, F93, N104, E144 of CaM-like protein is involved in the interaction with IQ1 motif. A maximum of 170 EF-hand proteins and 39 non-EF-hand proteins with Ca+2/metal binding motif were identified. Diverse proteins including enzyme, transcription, translation and large number of unknown proteins have one or more putative EF-hands. Phylogenetic analysis revealed seven major classes/groups that contain some families of proteins. Various domains that we identified in the EF-hand proteins (uncharacterized) would help in elucidating their functions. It is the first report of its kind where calcium binding loop sequences of EF-hand proteins were analyzed to decipher their calcium affinities. Variation in Ca+2-binding affinity of EF-hand CaBP could be further used to study the behaviour of these proteins. Our analyses postulated that Ca+2 is likely to be key player in Caenorhabditis cell signalling.Calcium (Ca Transient elevation of Ca+2 is perceived by two classes of proteins: Ca+2 sensors and Ca+2 buffers +2 with the Ca+2 sensors  leads to the conformational change, resulting in alteration of its activity or its ability to interact with other proteins or nucleic acids. Interaction of Ca+2 sensors with its target results in the modulation of target's function or activity 2+ buffers  are a smaller subset of the EF-hand protein family which does not undergo a significant conformational change on binding to free Ca2+2+ signal either by transmitting the signal throughout the cell or helping the cell in getting rid of the free Ca2+ from the cytoplasm All living cells are required to have a cross talk with the environment where they reside. This cellular communication is facilitated by a lot of messengers. Calcium (Ca+2 coordination loop that has 12 residues (10–21). The last part is helix F that is at C terminal and consists of 10–12 residues (19–29). Structurally the two helices  E and F are 90 degree to each other. Residue 1 is often Glu (E) and a Gly (G) at residue 15 is highly conserved, as is Ile (I) at residue 17. The name EF-hand is derived from the three-dimensional arrangement of helix, loop and helix, which gives an impression of the thumb, index and middle fingers of a hand In the EF-hand family of proteins the functional unit is a pair of EF-hand motifs +2 coordination loop at position 1, 3, 5, 7, 9, and 12. These residues offer Oxygen for Ca+2 binding. At position 12 of Ca+2 coordination loop generally glutamate is found that offers both its side-chain oxygen for Ca+2 coordination. Out of seven ligands used for the coordination with the Ca+2, six are given by twelve-residue loop  and seventh is the water molecule. Residues 1, 3, 5 and 12 offer side chain carboxy group for coordination with the Ca+2 and residue at 7 coordinates through backbone carbonyl group. Residue 9 forms link with Ca+2 through water bridge +2 binding constant in different CaBP and might be having some correlation with the sequence position at 1, 3, 5, 7, and 9.The canonical EF-hand motif, which has been most extensively studied, is highly conserved in the Ca+2 and affinity for target proteins In most of the cases functional unit of EF-hand proteins is a pair of EF-hand motifs and proteins with four EF-hands usually have one pair of EF-hands in one domain and the other pair in second domain. Calpain is an exception to EF-hand pairing rule. It comprises of two subunits with each subunit having five EF-hands. The unpaired EF-hand in subunits pair to form heterodimer C. elegans, may bind to IQ motifs of myosin. Myosins, neuronal growth proteins, voltage-gated channels and certain signalling molecules contain IQ motifs Saccharomyces cerevisiae (Myo2P) on CaM-like protein and studied three-dimensional structure of the complex between IQ1 and CaM (IQ1-CaM-like protein).CaM-like protein from Caenorhabditis genus including Calcineurin, CaM +2/CaM-dependent serine/threonine protein phosphatase +2C. elegans. Phosphodiesterase, phosphatase, kinases and myosins have been shown to bind CaM Caenorhabditis genome would give a deeper understanding of the biology of proteins. Functional and structural classification of EF-hand containing proteins can be a first step in the identification of CaBP that might be involved in cellular process. Keeping this in view we thoroughly analysed the Caenorhabditis genome for genes encoding proteins with EF-hand motifs.Several EF-hand proteins have been identified in C. elegans was carried out using Modeller program. For modeling of CaM-like protein, crystal structure of Drosophila melanogaster CaM was used as a template having PDB ID 2BKH|B +2 ions  was docked to the model CaM-like protein, keeping CaM-like protein as a fixed molecule, using GRAMM-X (C. elegans CaM-like protein might be regulating the myosin network in C. elegans.Comparative modeling of CaM-like protein (gi: 37699821) of  +2 ions . CaM-lik +2 ions . Overall GRAMM-X . Program GRAMM-X . H-bonds GRAMM-X . The mod+2 signalling pathways and Ca+2 sensors present in Caenorhabditis, identification of EF-hand using the Caenorhabditis genome sequences was undertaken. To identify EF-hand containing protein Caenorhabditis genome, protein sequences were retrieved from NCBI Protein database. Each protein sequence was then subjected to InterProScan Caenorhabditis genome, for EF hand containing proteins using sensitive sequence profile matching algorithms . The protein sequence of Caenorhabditis CaM was used as a template in these searches. Search procedures such as PSI-BLAST have been used at E-value cut off of 0.0001 Caenorhabditis was also done. A large number of EF-hand proteins were identified by these methods. We were able to decipher a substantial number of EF-hand proteins which were not even mentioned in the Uniprot. These protein sequences were given their respective NCBI gi number. While curating our data utmost care was taken to include mostly those proteins which were identified as EF-hand proteins by more than one database. As a result of this criteria used in the identification of EF-hand, there is remote possibility of selecting a false positive. Published literatures were also thoroughly searched for the report of EF-hand proteins in Caenorhabditis. All these search together lead to making of a comprehensive repository of EF-hand proteins which includes not only well known EF-hand proteins but also those that were not mentioned in other databases. In this analysis an attempt was made to not only to identify EF-hand proteins but also to identify non EF-hand Ca+2/metal binding proteins. Rationale for this approach was to identify all those proteins that may play an important role as Ca+2/metal sensors and transducing molecules due to the presence of a number of Ca+2/metal binding domains. We have been able to identify 170 EF-hand proteins and 39 non EF-hand proteins with Ca+2/metal binding ability  C. Briggsae has PLC domain. Phosphatidylinositol-specific PLC, a eukaryotic intracellular enzyme, plays an important role in signal transduction +2. The worm base and uniprot databases were also used to infer the functions for these proteins. Most of these functions have been proven through experiments like mutant phenotype etc. Functions range from development to neurological. Egg laying defective protein-8 (Q95X29) of C. elegans is involved in the embryo development, signalling pathways and regulation of mating behaviour There is also one class of proteins that has phosphatidylinositol-specific phospholipase C (PLC) profiles. CBR-PLC-1 (A8XRA5) The number of EF-hands in each protein varied from one to eight. Most of these proteins have a pair of EF-hands which establishes their functional relevance Caenorhabditis were scanned for the presence of Ca+2 dependent protein kinase having EF-hand but could not find out any. There is an evidence of Ca+2/CaM-dependent protein kinase II in one of the species of Caenorhabditis that is C. elegans but it does not have EF-hand Caenorhabditis genome. Troponin C is part of troponin I and troponin T complex which regulates muscle contraction. In one of the species of Caenorhabditis that is C. elegans, troponin C isoform 2 (Q09665) has four EF-hands but it has been found out that there are only two Ca+2 binding EF-hands Caenorhabditis genome which has three EF-hands. Myosin light chain regulatory protein (MLC-3), Spectrin protein Genomes of all the species of +2 binding domain EF-hand type.Some of the EF hand proteins have Eps15 homology domain Several identified domains like PLC Many of the EF hand proteins have Receptor-mediated endocytosis (RME) domain which has been shown to extend help in endocytosis process Caenorhabditis has so many three- to four-EF-hand-containing proteins with no other domain. These proteins are likely to bind only Ca+2 ions, as these do not have any extra sequences corresponding to other domain. These proteins's architecture looks quite similar to CaM. After binding Ca+2 these may undergo conformational change which makes them suitable for the activation of associated proteins.The unique aspect of the results is that the Caenorhabditis has a large number of EF-hand proteins non EF-hand proteins with Ca+2/metal binding motif. Are all these proteins functional? It is not possible to say anything about the functionality of a gene just on the basis of computational analysis. However, one of the ways to know the functionality of the genes is to check the expression profile of genes. If gene is expressed at both RNA and protein levels, there is a strong possibility that the gene is functional. Caenorhabditis.Data presented above states that Caenorhabditis. Some of these were found and summary of the results is shown in CaenorhabditisCaenorhabditis confirms the belief that one of the most important EF-hand proteins is CaM. Troponin C is present in C. remanei, C. brenneri, C. briggsae. There can be functional similarity in same protein from different species. Presence of identical proteins in more than species also helps in understanding the importance of that protein in the evolution. It also tells how essential that protein is for the functioning of that organism.Presence of these proteins was also checked in other species of Caenorhabditis. Ascaris, Loa, Dugesia, Trichinella, Dictyostelium and Onchocerca.Caenorhabditis. Many of the homologues found out in other organism are putative ones. So some inference about their function could be guessed from function of known homologue partner. Nearest homologue of C. elegans hypothetical protein M04F3.4 is Loa loa programmed cell death protein-6. Identity between them is around 77%. So the function of C. elegans hypothetical protein M04F3.4 could also be speculated on the basis of high degree of identity. In many cases same protein is closest homologue of more than one EF-hand proteins. Only difference among them is percentage of identity. Like C. briggsae AF16 HP CBG_02410 and C. brenneri troponin C closest homologue is Loa loa putative uncharacterized protein and percentage of identity is 44% and 95% respectively.CaM and CaM related protein homologues share a great deal of identity. Identity reaches as high as 92%. The reason for high percentage could be that CaM is highly conserved protein and very little change happens during the evolution of new species. In most of the cases, the closest homologue is present in worms. It satisfies the fact that on evolutionary ladder worms would be closest neighbour of Full length EF-hand protein sequences identified by InterProScan were aligned using MAFFT Phylogenetic analysis was carried out by MEGA5 using maximum likelihood method. A bootsrap consensus tree was constructed. The tree was used to identify group of proteins and closely related proteins, where total seven groups could be identified. In this group there is good number of hypothetical proteins . MLC-3 a+2 ions and binding of Ca+2 to EF-hands bring conformational change in the CaM which then allows CaM to interact with target proteins to modulate their activity or function. More than five CaM related proteins are present in Caenorhabditis and almost all of them are highly similar. There is some variation in the length and number of EF-hands of these proteins. CAL-4 has three EF-hands. CAL-1, CAL-2 and CAL-3 have four EF-hands. It also includes Troponin C which too has four EF-hands. As far as expression of these proteins are concerned not much is known except CAL-1 which is expressed in C. Elegans. The Ca+2 binding ability of these proteins have not been experimentally verified but amino acid sequence composition indicates that these should have strong Ca+2 affinity. CaM has also been shown to be involved in the activation of protein kinases Group II  is a col+2 binding ability has not been proven , Y48B6A.6c (two EF-hands) and CaBP F21A10.1 (three EF-hands). InterProScan shows that both can bind Ca+2.It contains Vitellogenin linked protein +2 dependent. CaBP1 which has 8 EF-hands probably plays a role in the signal transduction. ZK856.8 has three EF-hands and computationally it has been found out that it is involved in the signal trassduction and locomotion. W04D2.1 Caenorhabditis elegansAll cytoskeleton proteins fall in this group . Spectri+2. RME-1 isoform a, c, e and f have one EF-hand and on the basis of InterProScan we can say that it might bind Ca+2 ions. RME has Eps 15 homology domain. From electronic annotation it has been inferred that it has GTPase activity (GTP binding). ITSN-1 This group at the end of the Phylogenetic tree comprises of NCS-2, NCS-3 and different isoforms of RME. NCS-2 & NCS-3 are members of large family of NCS . Not all+2 binding attribute of an EF-hand sequence is governed by five amino acids of 12 amino acids Ca+2 binding loop sequence of EF-hand. If any mutation occurs in this loop sequence then there is an alteration in the Ca+2 binding affinity of the EF-hand. CaM has four EF-hands and all four EF-hands show high degree of Ca+2 binding affinity. Keeping this point in view we retrieved more than 10 CaM sequences of 10 different organisms. Ca+2 binding loop sequences of all CaMs were aligned and a consensus sequence was generated. More than 40 EF-hand sequences were aligned. This consensus sequence was compared with the Ca+2 binding loop sequence of EF-hand proteins and if there was 100% identity with consensus sequence at five critical positions of the loop that might be involved in the Ca+2 binding then it was considered as strong Ca+2 binding affinity. If the identity at five critical positions of the loop drops down to less than 70% then it was considered medium. And if the identity comes as less as 50% then it was regarded as low. As far as assignment of some number to the strong, medium or low Ca+2 binding constant is concerned, it was quiet difficult task in view of the fact that there was some variation among the Ca+2 binding constants of different CaM. This variation happens because there is some variation in the sequence also. To sort out the issue we tried to round about range value based on the Ca+2 binding constants of some CaM +2 binding constants. For strong, a range of 104 to 107 was considered; medium, less than 104; low less than 102.Ca+2 binding constants but one thing about this comparison is sure that the loop sequence which is 100% identical with the CaM consensus loop sequence is most probably going to have Ca+2 binding constant, comparable to that of CaM. Ca+2 binding loop sequence which is less than 100% identical to the CaM consensus loop sequence should have less Ca+2 binding constant in comparison to that of CaM. +2 binding constant of EF-hand CaBP. Here, out of the all studied proteins, Ca+2 binding affinity was significant in most of the cases and few were of medium affinity but we could not find any sequence having very low affinity.Though there could be lot of debate on the assignment of some numerical value to the strong, medium and low CaC. elegans and Drosophila melanogaster CaM are well conserved. The stable structure is used for docking with IQ1 motif. In this study it was found that A112, A39, E36 of CaM-like protein are important for strong hydrogen binding interaction with IQ1 motif. CaM-like protein residues F165, F169, L29, E33, F44, L57, M61, M96, M97, M108, G65, V115, F93, N104, and E144 play important role in interaction with IQ1 motif.CaM-like protein (gi: 37699821) model shares sequence homology of 65% with the template. The sequential and structural information between the CaM-like protein (gi: 37699821) from Caenorhabditis requires such a large number of Ca+2 sensors, unlike many other organisms? Some of the probable reasons would be that Ca+2, being major player in diverse functions and some of these being compartmentalized in different cellular locations, different CaBP may participate in different functions which are spatially and temporally separated. Caenorhabditis EF hand proteins control diverse array of cellular functions. MLC 4, Spectrin and alpha actinin are associated with cytoskeleton architecture Caenorhabditis+2 is involved.It is not clear why +2 signalling is enhanced due to the existence of many families of proteins. More than one isoform of CaM, NCS and RME are present in Caenorhabditis. The regulation of expression and kinetics of interaction of these isoforms with different proteins makes cell signalling processes further complex. The variation in the Ca+2 binding affinity adds a new dimension to the regulation of CaBP for fast association and fast dissociation is required for the switching on and off of CaBP. In summary, Caenorhabditis exhibits unusual array of Ca+2 sensors, indicating a complex network of Ca+2 signaling pathways. Further characterization of the CaBP is required for complete understanding of the roles of Ca+2 and Ca+2 signalling network.Complexity of CaC. elegans CaM-like protein (gi: 37699821) was retrieved from Swiss-Prot database Drosophila melanogaster CaM (2BKH|B) available at PDB was used as template for modelling. Sequence alignment between the model sequence and template was done with ClustalW C. elegans CaM-like protein was built by Modeller Amino acids sequence of Caenorhabditis genome database (http://www.ncbi.nlm. nih.gov). Each sequence was used for BLAST sequence (http://www.ncbi.nlm.nih.gov/BLAST) similarity search against Caenorhabditis genome database. InterProScan was extensively used to look for the presence of EF-hands and other domain in these proteins Amino acid sequences of all annotated CaBP and EF-hand proteins were retrieved from http://www.pfam.wustl.edu/hmmsearch.shtml), UniProt (http://www.uniprot.org) and WORM database were extensively searched for the EF-hand proteins and any other information related to it. Nearest homologues in species other than Caenorhabditis were also collected using BLAST.A number of search tools such as reverse BLAST, PSI-BLAST, CD-search, HMM search and other sequence searching algorithm was also used to identify maximum number of EF-hands. Other databases like Pfam  The phylogenetic tree and branch support values were estimated using Maximium Likelihood (ML) methodology of phylogenetic reconstruction using MEGA 5 +2 binding constants, CaM sequences of 10 different organisms were retrieved from swiss prot database. Ca+2 binding loop sequences of all CaM were aligned and a consensus sequence was generated. More than 40 EF-hand sequences were aligned. This consensus sequence was compared with the Ca+2 binding loop sequences of EF-hand proteins.For the prediction of CaTable S1Caenorhabditis.EF-hand containing proteins in (PDF)Click here for additional data file.Table S2Caenorhabditis.Non EF-hand with calcium/metal binding motif proteins in (PDF)Click here for additional data file.Table S3Caenorhabditis genus.Nearest homologue of EF-hand Proteins in organism other than (PDF)Click here for additional data file.Table S4Calcium binding constant of EF-hand CaBP.(PDF)Click here for additional data file.


JAM-C is a junctional adhesion molecule, enriched at tight junctions on endothelial and epithelial cells, and also localized to Schwann cells at junctions between adjoining myelin end loops. The role of JAM-C following peripheral nerve injury (PNI) is currently unknown. We examined the localization of JAM-C after sciatic nerve crush injury in adult rats. JAM-C immunoreactivity was present in paranodes and incisures in sham surgery control nerve, but distal to the crush injury significantly decreased at three and 14 days. JAM-C was re-expressed at 28 days and, by 56 days, was significantly increased in the distal nerve compared to controls. In a 7-mm length of sciatic nerve sampled distal to the crush site, the densities of JAM-C immunoreactive paranodes increased in the distal direction. Conversely, the densities of JAM-C immunoreactive incisures were highest immediately distal to the crush site and decreased in the more distal direction. Further analysis revealed a strong correlation between JAM-C localization and remyelination. Fifty-six days after crush injury, greater densities of JAM-C paranodes were seen compared to the nodal marker jacalin, suggesting that paranodal JAM-C precedes node formation. Our data are the first to demonstrate a potential role of JAM-C in remyelination after PNI. JAM-C belongs to the immunoglobulin superfamily class of junctional adhesion molecules, composed of JAM-A, -B, -C, ESAM, CAR, and JAM4. It is characterized by two immunoglobulin folds in the extracellular domain , and is JAM-C deficiency in peripheral nerves alters the integrity of the myelin sheath and consequently causes defective nerve conduction , indicatIn the peripheral nervous system (PNS), myelinating Schwann cells wrap around axons, organizing the axonal membrane into distinct domains . EfficieFollowing crush injury, nerve fibers distal to the lesion degenerate in a process known as Wallerian degeneration. Schwann cells dedifferentiate, reversing molecular expression from a mature myelinating state to an immature one , and expAdult male Wistar rats (225–250 g) were deeply anesthetized in a fume box using 5% isofluorane , in addition to a mixture of oxygen and nitrous oxide (1:1 ratio), at a flow rate of 750–1000 mL/min. Subsequent anesthesia throughout the procedure was maintained using 2–2.5% isofluorane, with oxygen and nitrous oxide at an unchanged ratio, delivered through a nose-piece. The left sciatic nerve was exposed at mid-thigh level. A crush injury was induced to the sciatic nerve for 15 sec, using a pair of microforceps. After injury, the site of crush was marked with sterilized ink. Then the overlying muscle and skin were sutured, and the animals were returned to warm cages to recover from anesthesia. All experimental protocols were approved by the local ethics committee and in accordance with the U.K. Home Office regulations .n= 4 per time point), animals were overdosed with sodium pentobarbital  and perfused via the ascending aorta with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. A piece of the left sciatic nerve was dissected out, spanning from at least 5 mm proximal to the crush site to about 10 mm distal to the site, where the sciatic nerve starts to branch. The tissue was postfixed in the same fixative for 2 h at room temperature (RT), and then cryoprotected in 30% sucrose solution in 0.01 M phosphate buffered saline (PBS) overnight at 4°C. All sciatic nerves were then embedded in OCT medium  at –20°C and stored at –80°C until further processing. Four additional control rats, which had sham operations, were also processed in this way. Serial longitudinal sections of 8-μm thickness were cut using a cryostat, and consecutive sections processed for immunohistochemistry.At three, 14, 28, or 56 days postsurgery , rabbit anti-JAM-C polyclonal anti-body (1:800∼1:1500). Lectin staining was performed using the fluorescein-labeled jackfruit agglutinin . Appropriate secondary antibodies conjugated with Alexa Fluor dyes were purchased from Invitrogen  as follows: goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 586 . Hoeschst 33342  was used to reveal cell nuclei. All primary and secondary antibodies as well as the normal goat serum (NGS) were diluted in PBS containing 0.2% Triton X-100 and 0.1% sodium azide. The PBS wash (3 × 10 min) was a standard routine after incubation with a primary or secondary antibody. All incubation was carried out in a humidified chamber at RT.The following primary antibodies were used: mouse anti-200-kDa neurofilament , mouse anti-P0 , mouse anti-S100 , mouse antimyelin-associated glycoprotein , mouse anti-pan NaV, or anti-MAG antibody overnight. After 2 h incubation with anti-mouse secondary antibody, followed by PBS washes, sections were then counterstained with Hoeschst for 2 min. The slides were then rinsed with distilled water before final mounting in PBS glycerol (1:8). For P0 and JAM-C double labeling, sections were first treated with ice-cold methanol (–20°C) for 10 min. After incubation with the P0 primary antibody followed by Alexa Fluor 568, sections were incubated with the JAM-C primary antibody, followed by incubation with Alexa Fluor 488. For double labeling after lectin staining, NGS was applied for 1 h after 15-min jacalin application, and was then followed by primary and secondary antibodies using routine methods as described above. Specificity was confirmed in controls by incubating with secondary antibodies after omission of the primary antibodies. The characteristics of the anti-JAM-C antibody have been reported previously  using appropriate filter blocks . Images were taken using a Hamamatsu C4742–95 digital camera  and the Leica QWin program . Figures were prepared using Adobe Photoshop CS2.In the injured rat sciatic nerve, the crush site was relatively easy to identify based on the marked reductions of P0, N52, or JAM-C staining. P0 images were taken at ×20 objective magnification at three areas: namely 1.4-, 4.0-, and 6.6 mm distal to the crush site. Images were also taken 1.4 mm proximal to the crush site.The Leica QWin software was used to quantify the P0 immunostained myelin by converting the camera image into a binary image of the P0 labeling. Three measuring frames of identical size (640 μm × 640 μm) were then randomly applied onto each image, and the percentage of the measuring frames covered by this binary image was determined. The mean of these three measures was then determined for each area per animal. Regions were analyzed from at least three sections per animal. In some distal nerve areas, myelin debris was manually excluded in Photoshop and then the above quantification performed. This method of analysis was chosen because P0 labeling was too extensive to allow for unambiguous identification of individual axons and myelin. However the analysis does not distinguish between decreased P0 labeling due to thin myelin, and decreased P0 labeling due to decreased space occupied by myelinated nerve fibers.V immunoreactive nodes and MAG immunoreactive incisures were counted in a similar way as for JAM-C. The results were expressed as densities . Regions were analyzed from at least six sections per animal.For analysis of JAM-C localization, images were taken at ×40 objective magnification at each location as described above. In Photoshop, a counting grid was placed over each image and the total number of JAM-C immunoreactive paranodes or incisures was counted. Jacalin immunoreactive or NaP < 0.05 was considered statistically significant.All results are expressed as mean ± standard errors of the means (SEM). One-way, two-way, or repeated measures of analysis of variance (ANOVA) were used when appropriate. The Tukey–Kramer multicomparison adjustment was used as the post-hoc test to calculate the significance levels. V) and with a marker of Schmidt–Lantermann incisures (MAG) confirmed that JAM-C is concentrated in paranodal regions of nerves , immunolabeling followed by quantitative analysis of paranodes and incisures was performed spatially in the near, mid-, and far-most distal regions  along the distal nerve. Additionally, this localization was examined temporally at various time points; namely three, 14, 28, and 56 days after nerve crush. These time points were selected as covering both the degeneration stage (three days) and the remyelination process, which is known to begin within two weeks of the onset of axonal regeneration in rats .2 vs. 115 ± 4/mm2; P= 0.004). The intermediate and far-distal regions (4.0 and 6.6 mm) showed an almost complete deterioration of JAM-C immunoreactive paranodes at both three and 14 days (At three (not illustrated) and 14 days  after in 14 days , and 3a. 14 days  and 3a. P < 0.05). In contrast to the loss of JAM-C immunoreactivity following earlier time points, we observed a substantial increase of JAM-C paranodal immunoreactivity at 56 days in the distal nerve as compared with either the controls or the proximal region of the nerve . However, paranodes appeared narrower and shorter in size compared to uninjured nerves, and this was confirmed by quantitative analysis . JAM-C ihe nerve . The parhe nerve , similarhe nerve . At 1.4 he nerve . Meanwhi2; P < 0.05; JAM-C immunoreactive incisures were examined in a similar spatiotemporal manner as JAM-C immunoreactive paranodes. At three (not illustrated) and 14 days  after in2 vs. 986 ± 30 incisures/mm2; P < 0.05).JAM-C immunoreactive incisures appeared to show numerical recovery by 28 days  after in2 in the near-distal region compared to 1114 ± 65 JAM-C immunoreactive incisures/mm2 in the far-distal region , whereas in the far-distal region there was a 91% reduction in P0 localization . This spatial pattern of localization closely resembles that observed with JAM-C immunostaining.A reduced level of P0 staining was observed at 14 days following injury, with the continuous myelin layers having disappeared distal to the crush site . A dis-oP < 0.05). By 56 days . However, there yet remained a 33% decrease in the near-distal nerve, with a 62% decrease in the far-distal nerve .At each time point, the density of both JAM-C immuno-reactive paranodes and incisures appeared to follow the course of myelination. A Spearmann's rank test  confirmeV , and with MAG, a marker for incisures. In separate experiments, jacalin was shown to be expressed at nodes of Ranvier, as observed through double labeling with the well-established marker, pan-NaV . MoreoveP < 0.05; P < 0.05). Throughout the injured nerve, we hardly ever observed jacalin nodes without JAM-C paranodes.Our results in controls showed that not all of the paranodal regions were positive for JAM-C, with further quantification showing only ∼80% of the total jacalin immunoreactive nodal regions were surrounded by JAM-C immunoreactive paranodes , the proximo-distal gradient of JAM-C localization is consistent with regeneration proceeding in the anterograde direction, and Schwann cells re-expressing JAM-C as they mature into a myelinating phenotype. It is well known that Schwann cells classically undergo phenotypic modulation during development and in response to injury . During At 56 days postinjury, significantly increased numbers of JAM-C immunoreactive paranodes were present in the region distal to the crush site, with JAM-C immunoreactive paranodal densities highest in the far-most distal region; almost 2.5-fold compared to numbers in uninjured sciatic nerve. This trend may be explained by abnormally short internodal distances, thus resulting in increased numbers of JAM-C immunoreactive paranodes. Abnormally short internodal distances have been implicated in causing a conduction velocity lag in regenerated axons, as they are formed by more than a threefold increase during Schwann cell proliferation in the distal nerve stump . These dIn contrast, the JAM-C immunoreactive incisural densities decreased proximo-distally, with the highest numbers in the near-distal region. As incisures subserve a role to maintain myelin sheath integrity, it is likely that the increased numbers are present to help provide stability between the various myelin sheath layers. Their smaller size, postinjury, may be related to thin myelin during remyelination. The localization of JAM-C is specific to noncompact myelin; that is, at the incisures and paranodes, where a wide variety of specialized junctions exists, including gap, adherens, and tight junctions. These regions are believed to be critical for signaling, transport of small metabolites, and maintenance of myelin structure . JAM-C mFrom our measurements of P0 myelin density, at the most distal region at 56 days, myelination had not yet reached the levels of controls. This implies that the increased JAM-C localization may possibly be due to the remyelinating nerve still being present in a remodeling stage. In rats, 56 days remains a relatively long period of time for recovery after injury, and it is potentially possible that a chronic pathological state may have been reached, in which there is a disproportionate ratio of paranodes to incisures. Consistent with our results, walking track analysis has shown that even after eight weeks, recovery after sciatic nerve crush in rats is only 40% of that of controls .V at different survival times post-injury. However, another interesting possibility is that paranodal JAM-C is expressed ahead of the nodal structure during the development of the paranodal–nodal region. Indeed, in the CNS, paranodal proteins, including neurofascin, appear to cluster before node formation and facilitate ion-channel clustering , we showed that JAM-C localization correlated highly significantly with the degree of P0 myelin localization, with greater JAM-C localization associated with increased remyelination. We have also shown, as discussed above, that JAM-C localization in paranodes may precede node formation. Our results indicate that JAM-C may play an important role in node formation and/or myelin localization, and it would be interesting to search for ligands that interact with JAM-C, to determine the mechanisms that underlie Schwann cell to Schwann cell JAM-C signaling. However, further studies are required to establish a direct causal relationship.


Pinprick perception, 128-Hz tuning fork vibration detection, ankle reflexes, vibration perception threshold (VPT) with a biothesiometer or similar instrument, and four site 10g monofilament assessment are all currently recommended methods for screening for diabetic peripheral neuropathy (DPN). However, there is limited research investigating the reliability of these tests. The aim of this study was to determine the inter- and intra-tester reliability of the five neurological tests currently recommended for screening for DPN.All five recommended neurological examinations were performed by three clinicians on people with diabetes to determine inter-tester reliability. The tests were repeated by the same clinicians seven days later to determine intra-tester reliability.Fifty participants with diabetes were recruited to this study, 44 returned for the re-test. The intra-tester reliability of the VPT with the neurothesiometer was substantial (К: 0.52-0.78), monofilament test was moderate (К: 0.34-0.67), and the pinprick and ankle reflex examinations was fair (К: 0.15-0.32 and 0.09-0.62 respectively). The inter-tester reliability of the neurothesiometer and monofilament was substantial (К: 0.61 respectively), the pinprick examination moderate (К: 0.52) and ankle reflex examination slight (К: 0.12). The reliability of the vibration perception examination using the 128-Hz tuning fork could not be calculated due to all participants recording an abnormal result.For the purposes of clinical screening and ongoing monitoring of DPN the four site 10g monofilament test and the vibration perception threshold examination using the neurothesiometer are the most reliable of the recommended screening examination for people with diabetic peripheral neuropathy (DPN).


To combine global cardiac function imaging with compressed sensing (CS) in order to reduce scan time and to validate this technique in normal mouse hearts and in a murine model of chronic myocardial infarction.To determine the maximally achievable acceleration factor, fully acquired cine data, obtained in sham and chronically infarcted (MI) mouse hearts were 2–4-fold undersampled retrospectively, followed by CS reconstruction and blinded image segmentation. Subsequently, dedicated CS sampling schemes were implemented at a preclinical 9.4 T magnetic resonance imaging (MRI) system, and 2- and 3-fold undersampled cine data were acquired in normal mouse hearts with high temporal and spatial resolution.The retrospective analysis demonstrated that an undersampling factor of three is feasible without impairing accuracy of cardiac functional parameters. Dedicated CS sampling schemes applied prospectively to normal mouse hearts yielded comparable left-ventricular functional parameters, and intra- and interobserver variability between fully and 3-fold undersampled data.This study introduces and validates an alternative means to speed up experimental cine-MRI without the need for expensive hardware. J. Magn. Reson. Imaging 2011. © 2011 Wiley Periodicals, Inc. MULTIFRAME MAGNETIC RESONANCE IMAGING (cine-MRI) has become an established tool to assess ventricular volumes and mass in hearts of genetically and surgically modified mice and rats, allowing for a noninvasive characterization of global cardiac function in these models of human cardiac disease. Cine-MRI exams in mice are commonly performed at dedicated small-bore MRI systems equipped with ultrahigh magnetic fields ≥7T, using surface- ,2, volumk-space followed by a nonlinear reconstruction algorithm (see below).Compressed sensing (CS)  is a tecFew studies have reported on the application of CS in cardiac MRI ,19, but 2, intubated, and ventilated with a tidal volume 250 μL and respiratory rate 150/min . A left thoracotomy was performed in the fourth intercostal space. An intramyocardial suture was placed 1–2 mm below the atrio-ventricular groove with an atraumatic needle and a 6–0 polyethylene suture. Mice were given subcutaneous buprenorphine (0.8 mg/kg) for pain relief. In the validation stage of the project the chronic myocardial infarction group together with matching sham-operated mice  were scanned with a standard cine-MRI protocol  at least 1 week prior to the MR examination or surgical procedure to allow naturalization to new surroundings. The mice were kept under controlled conditions for temperature, humidity, and light, with chow and water available ad libitum. Five mice (25.9 ± 0.7 g) were subjected to permanent ligation of the left anterior descending coronary artery to generate a chronic myocardial infarction as previously described . In brieTo prepare the mice for cine-MRI, anesthesia was induced in an anesthetic chamber using 4% isoflurane in 100% oxygen. Animals were then positioned prone on a warm air blanket (37°C) in the mouse cradle and maintained at 1.5%–2% isoflurane at 2 L/min oxygen flow throughout the MRI experiments. Cardiac and respiratory signals were continuously monitored using an in-house developed ECG and respiratory gating device .Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 (HMSO) and to institutional guidelines.All investigations conformed to Home Office 2, matrix size 256 × 256, TE/TR = 1.7/4.6 msec, 15° sinc excitation pulse, number of averages NT = 2. The number of frames per cardiac cycle was determined by the heart rate and the number of phase-encoding steps per respiration cycle—acquired in a segmented fashion (n = 5) using dedicated undersampling patterns for phase-encoding direction as described below.MRI experiments were carried out on a 9.4T (400 MHz) MR system  comprising a horizontal magnet (bore size 210 mm), a VNMRS Direct Drive console, an actively shielded gradient system , and a quadrature-driven birdcage resonator (id 33 mm). After positioning the mice in the magnet with the heart in the center, and scouting for long- and short-axis orientation of the heart using a double-gated, segmented gradient-echo (GE) sequence, shimming and pulse calibration were performed automatically prior to each experiment. Eight to nine contiguous, 1-mm-thick slices were then acquired in short-axis orientation covering the entire heart using an ECG-triggered and respiratory gated multiframe sequence with steady-state maintenance during respiration A from eak-space data for each cine-frame in every slice of the sham and the MI group were retrospectively undersampled in one dimension by a factor of 2, 2.5, 3, and 4, respectively. The sampling scheme was changed for each frame and represented random acquisitions with an additional weight to the center of k-space. The patterns for the whole cycle of all acquired cine-frames approximated a Gaussian distribution to realize dense or nearly dense sampling of the low spatial frequency information, which has proven to be beneficial for a successful CS reconstruction  represents a useful tool to determine the coherence of the artifacts caused by a dedicated (random) sampling pattern in st limit . Specifik-space as applied in the retrospective studies. The patterns, which were causing the smallest value for the sidelobe to peak ratio  were stored and used for the measurements. Additionally, it was ensured that every phase encoding step was measured at least once over the whole cycle of measurements. k-space weighting can easily be seen.A simulation step was performed prior to the acquisition of real undersampled datasets to determine a suitable series of sampling patterns as a function of the number of timeframes  in the cine-train per slice, and the degree of undersampling . In all, 200 patterns were created per given number of frames in the cine-train and the according PSFs were determined for the undersampled dimension. The acceleration was limited to the phase-encoding direction and featured random schemes with an additional Gaussian weight to the center of 2) were loaded into Amira 4.1  and segmented as described before  and interobserver  variability were determined in fully and 3-fold accelerated acquisitions in the control group. To test differences between intra- and interobserver variabilities for the two acquisition techniques, Levene's test for equality of variances was used in SPSS.A general linear model analysis for repeated measures was used  to compare fully and undersampled data obtained in all three groups  individually. The Bonferroni correction was applied as a post-hoc comparison of the protocols. A P = 0.002 and P < 0.001), and for EF and SV in chronically infarcted hearts . Post-hoc analysis indicated nominal evidence that 4-fold undersampled data were significantly different from the other groups, but this was no longer the case after correcting for multiple testing (Bonferroni), except for SV in sham animals. For this parameter, statistically significant differences were found between 4- and 2.5-fold undersampled data (P = 0.03), 4- and 2-fold undersampled data (P = 0.02), and 4-fold undersampled and fully sampled data (P = 0.008), respectively. The same trend to underestimate SV was observed in infarcted mice, but failed to reach statistical significance, presumably due to higher variability between individuals. Infarct sizes, which were measured in fully sampled datasets only as described in .P = 0.018); however, this represents a difference in volume of only 2.1 μL. Furthermore, comparable inter- and intraobserver variabilities were obtained in these datasets (n = 5).datasets . Levene'13C-chemical shift imaging of murine cancer models  as proposed by Lustig et al  hardware requirements.


Although changes in these regions are found in carriers of the s allele and/or in depressed patients, evidence for a specific genotype × developmental stress effect on brain structure and function is limited. Here, we investigated the effect of repeated stress exposure during adolescence in mice with partial knockout of the 5-HTT gene (HET) vs. wildtype (WT) on early-adulthood behavioral measures and brain structure [using magnetic resonance imaging (MRI)] relevant to human major depression. Behaviorally, adolescent stress (AS) increased anxiety and decreased activity and did so to a similar degree in HET and WT. In a probabilistic reversal learning task, HET-AS mice achieved fewer reversals than did HET-No-AS mice. 5-HTT genotype and AS were without effect on corticosterone stress response. In terms of structural brain differences, AS reduced the volume of two long-range white matter tracts, the optic tract (OT) and the cerebral peduncle (CP), in WT mice specifically. In a region-of-interest analysis, AS was associated with increased HC volume and HET genotype with a decreased frontal lobe volume. In conclusion, we found that 5-HTT and AS genotype exerted long-term effects on behavior and development of brain regions relevant to human depression.In humans, exposure to stress during development is associated with structural and functional alterations of the prefrontal cortex (PFC), amygdala (AMY), and hippocampus (HC) and their circuits of connectivity, and with an increased risk for developing major depressive disorder particularly in carriers of the short ( We hypothesized that: (1) AS and HET genotype would lead singly and additively to long-term increased anxiety in behavioral tests thereof; (2) AS would lead to reduced performance in a probabilistic reversal learning task in both WT and HET mice; (3) AS and HET genotype would lead singly and additively to reduced volumes of the frontal lobe, AMY, and HC.ad libitum food and water. Behavioral training and testing was carried out under dimmed light conditions during the dark phase. All procedures were conducted under a permit for animal experimentation issued by the Veterinary Office, Zurich, Switzerland in accordance with the Animal Protection Act (1978) Switzerland. All efforts were made to minimize the number of mice used and any extraneous stress of those mice that were used.Male and female mice of a 5-HTT null mutant strain on a C57BL/6J background (>20 backcross generations) were transferred from the University of Würzburg  group and studied in one of two experiments. The AS/No-AS manipulation was performed between postnatal days (PND) 28 and 47, and mice were studied in readout tests across late adolescence and early adulthood.Experiment 1 included 44 mice  that were tested at PND 54–59 in the novelty induced hypophagia test and 1 day later in the elevated plus maze test, to assess anxiety. At PND 80–84, a serial blood sampling procedure was conducted to assess reactivity of the hypothalamic-pituitary-adrenal-axis. Between PND 92 and 150, mice were trained and tested in a probabilistic reversal learning task.Experiment 2 included 54 mice that were tested at PND 64–69 in the open field to assess activity and anxiety. At PND 80–83, the brains were fixed and studied using structural MRI. Three subjects were excluded due to a problem with the realignment procedure, so that the final sample size was HET-No-AS = 14; HET-AS = 12; WT-No-AS = 12; WT-AS = 13.The time lines of experiments 1 and 2 are detailed in Figure The stress procedure consisted of nine sessions of electro-shock exposure (S1–S9). S1 and S9 were always conducted at PND 28 and 47, respectively; otherwise, three sessions were conducted per week and sessions S2–S8 were distributed regularly between PND 29 and 46, with one session per day and two consecutive daily sessions maximum. Each session consisted of 24 electro-shocks of variable duration , administered on a variable interval schedule . The electro-shock intensity (mA) increased across S1–S9 to reduce habituation to the stress procedure: 0.1 (S1), 0.15 (S2), 0.2 (S3), 0.25 (S4 and S5), and 0.3 (S6–S9). Mice in the No-AS group were placed in the environment with an electro-shock intensity of 0. The total locomotor distance (arbitrary units) and total freezing duration during the intervals between electro-shocks were measured.Stressor sessions were conducted using a fully-automated apparatus  as described in . The apparatus comprised two closed arms enclosed by walls 15(H) cm, two open arms (OA) 30(L) × 5(W) cm and a center (5 × 5 cm) quadrant, elevated on legs [50(H) cm]. The EPM test was conducted. The mouse was placed on the center of the maze facing an open arm. Entries into and time spent on the open and closed arms were measured during 5 min using a video-tracking system . Total arm entries and distance moved were used as indices of locomotion; the ratio of OA entries to total arm entries and the ratio of time spent in the OA to total time spent on arms, were used as indices of anxiety.The test was conducted between PND 64 and 69 in mice that were subsequently studied in the structural brain analysis (Experiment 2). The OF measured 50 × 50 × 30(H) cm; the test was conducted under high illumination (50 lux in the center of the arena). After overnight habituation to the test room, mice were placed into the center of the OF. Mouse location and movement were monitored using a video-tracking system . The OF was divided virtually into center (30 × 30 cm) and periphery. The OF test duration was 30 min and data were analyzed in 5-min bins. Data for one mouse (WT-No-AS) could not be used due to a technical problem. Total distance moved was used as an index of activity and percent time spent in the center was used as a measure of anxiety i.e., greater avoidance of center equals greater anxiety.At PND 80–84, SBS was performed as described in .The test conditions were a maximum of 60 rewards or 30 min and on each trial the mouse could make an operant response to the left or right nose-poke port, with one nose-poke set to correct (sucrose pellet) and one set to incorrect/punish (no pellet + 5 s time out). After eight consecutive correct trials the nose-port—outcome contingency was reversed. In addition to the reversal schedule , a proportion of correct responses were punished. The probability of punished correct responses was set to 0.1 or 0.2. Additional test settings were that the first correct response per session was never punished and the maximum number of consecutive punished correct responses was set to 2. The evidence suggests that at 0.1 punished correct responses mice maintain accurate reward expectancy and punishment expectancy  with stress exposure  and 5-HTT genotype  as between subject factors. The AS procedure, OF and PRL data, as well as plasma corticosterone titers and body weight, were analyzed using repeated measures ANOVA (RM-ANOVA) with stress exposure , and genotype  as between subject factors and session or interval as within subject factors. For the NIH data, a survival analysis according to the Kaplan-Meier and log-rank (Mantel-Cox) tests was conducted; Cox proportional hazards regression model was used to compare the relative influences of stress exposure  and genotype  on latency to eat.Statistical analyses were conducted using StatView 5.0.1 . All data are reported as mean ± standard error of the mean (s.e.m.), with significance set at At PND 80–83, mice were anesthetized with pentobarbital-Na injected i.p. and transcardially perfused at a rate of 1 ml/min with 30 ml phosphate-buffered saline  containing 2 mM ProHance® contrast agent solution at room temperature (25°C), followed by 30 ml ice-cold 4% paraformaldehyde in PBS/2 mM ProHance®. The heads were removed and the skin, lower jaw, ears and cartilaginous nose tip were removed from the skull. The brains in the remaining skull structures were fixed in 4% paraformaldehyde containing 2 mM ProHanceat 4°C for 24 h. The skulls were then transferred to PBS with 0.02% sodium azide and 2 mM ProHance® solution at 4°C until they were scanned  was used to acquire anatomical images. A custom-built 16-coil solenoid array was used to image 16 samples concurrently  = 22.5, p < 0.007]; a posteriori pair-wise comparisons demonstrated that body weight (g) was higher in WT-No-AS compared to HET-No-AS  and to HET-AS mice . However, there was no significant difference when the mice were tested in the OF (PND 64–69) and thereafter.In Experiment 1, mice showed no significant difference in body weight between groups at any age. In Experiment 2, there was a significant genotype × AS interaction effect prior to AS group allocation  and in WT mice there was no AS effect.In Experiment 1, there was a significant AS × interval interaction for % time freezing  and WT-AS mice . For total number of entries there was a significant genotype effect ; WT mice made more total arm entries than HET mice . For total distance moved there was a significant main effect of genotype , with WT mice traveling a greater distance than HET mice (data not shown).For % time on OA, there was a significant genotype × AS interaction  was due to the monotonic decrease in activity by all groups across test intervals. The % distance moved in the center increased over time as indicated by a main effect of interval . There was no significant effect involving AS or genotype on % distance moved in the center.For total distance moved in the OF, there was a main effect of AS  which was due to a lower plasma CORT titers at 0 min relative to 20, 60, and 120 min . There was also no significant effect of AS or genotype on delta CORT values for time at 20 minus 120 min (p > 0.3), indicating a similar recovery of CORT concentrations to basal levels across groups.For plasma corticosterone (CORT) titers there was a significant main effect of sample : a posteriori pair-wise comparisons demonstrated that HET-AS mice completed fewer reversals than HET-No-AS mice . For session duration there was a significant genotype × AS interaction : a posteriori pair-wise comparisons demonstrated that session duration was longer in HET-AS mice than HET-No-AS mice  and no significant pair-wise differences involving WT mice. This potential indication of reduced task motivation was investigated using additional measures: for mean post-reinforcement latency (s) there was a borderline non-significant AS × genotype interaction : HET-AS mice (6.1 ± 0.8) had a longer post-reinforcement latency than HET-No-AS mice . In terms of % trials with a long post-reinforcement latency , there was a significant genotype × AS interaction : HET-AS mice had a higher score for this measure (4.4 ± 1.4 %) compared to HET-No-AS mice . As given in Table Table F, 37 = 4.p > 0.9, genotype p > 0.7, genotype × AS: p > 0.9). Analysis of the normalized volumes of all 62 structures showed that AS led to a significant volume decrease in two white matter regions, the optic tract (OT) at 5% FDR and the cerebral peduncle (CP) at 10% FDR. To assess whether this effect was associated with a genotype × AS interaction, separate RM-ANOVAs were conducted on the normalized volumes of the OT and CP. A significant interaction was found for the OT  and to HET-AS mice . The interactions did not survive correction for multiple comparisons in the whole brain analysis.Total brain volume showed no significant differences between groups  = 7.93, p < 0.008, Figure F = 6.8, p < 0.02, Table F = 4.4, p < 0.05] and not in the left hemisphere (p > 0.1), although this result did not survive Bonferroni correction for multiple comparison. The voxel-wise analysis is reported in Supplementary Figure p > 0.2). The right AMY was greater in volume than the left AMY [F = 181.6, p < 0.0001, Table For the normalized frontal lobe volume [based on the population-specific atlas  and locomotion (OF test). In WT and HET mice, AS led to increased latency to eat a palatable food in the NIH test and reduced distanced traveled in the OF test, indicating development of increased anxiety and reduced locomotor activity, respectively. In HET mice specifically, AS led to decreased percentage of entries on the open arms indicating development of increased anxiety. Hyper-anxiety endophenotypes have been clearly reported in 5-HTT KO mice . However, in HET mice increased anxiety has not been found consistently, even in adulthood. For example, in a previous study investigating the effects of prenatal stress, HET mice showed increased time spent in the open arm suggestive of reduced anxiety  compared to HET-No-AS mice, with WT mice exhibiting intermediate performance between the two HET groups. The increased session duration of HET-AS mice was associated with an increased number of trials with long post-reinforcement latency to make an operant response after delivery of the previous reward. This relatively high number of trials with long post-reinforcement latency likely contributed to the lower number of reversals completed: such long intervals put greater demand on working memory and have a higher probability of leading to a reward-shift error on the next trial; if such an interval occurs and leads to an error when the mouse is close to the reversal criterion  then this will have a higher impact on number of reversals completed than the more common reward-shift errors made just after reversal. There are two possible explanations for these long post-reinforcement latency trials. Firstly, HET-AS mice could have been less motivated to perform the task. However, on two other motivation measures, namely the number of feeder responses and the latency to collect the reward, no differences were found. Second, HET-AS mice could have been more sensitive to the high response-reinforcement uncertainty associated with the PRL task. Although the long post-reinforcement latency did not occur disproportionately often after false negative feedback or an incorrect response, reward expectancy is the major determinant for the number of reversals completed , intolerance of uncertainty is found in major depression and anxiety disorders  trials in an emotional Go/No-Go task compared to s/s carriers not exposed to childhood adversity . In contrast to our hypothesis, we did not detect any AS affect on the frontal lobe.In humans and other primates, the In addition, the whole brain analysis showed a genotype × AS interaction in two white matter tracts, the CP and OT. In WT mice specifically, AS decreased CP and OT volumes, whereas in HET mice AS had no significant effect. Data in humans and animals consistently demonstrate effects of developmental stress on white matter structures, including the corpus callosum  conferred increased resilience to further stress exposure. Repeated exposure to predator stress in adolescence, for example, has been shown to increase resilience rather than anxiety- and depression-related behaviors if rats were housed with conspecifics during the stress exposure (Kendig et al., In conclusion, repeated stress exposure during adolescence had long term effects on behavior and structural brain development. AS led to increased anxiety and decreased locomotor activity, in the absence of effects on HPA reactivity. In addition, in HET mice specifically AS impaired behavior in the PRL task: HET-AS mice showed a decreased number of reversal completed and an increased post reinforcement latency compared to HET-No-AS mice, suggesting that HET mice exposed to AS develop increased sensitivity to environmental uncertainty. In terms of structural brain differences, AS was associated with decreased volume of the long-range white matter tracts OT and CP in WT mice specifically. In addition, the region-of-interest MRI analysis showed that AS was associated with an increased HC volume and the HET genotype with a decreased frontal lobe volume. Future studies investigating the functional significance of these structural findings are warranted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.